ALPHA-HELIX DIPOLES AND CATALYSIS - ABSORPTION AND RAMAN-SPECTROSCOPIC STUDIES OF ACYL CYSTEINE PROTEASES

Raman and absorption spectroscopic data are combined with the deacylat ion rate constants for a series of acyl cysteine proteases to provide insight into the role of alpha-helix dipoles in rate acceleration. The Raman spectra, obtained by Raman difference Spectroscopy, of (5-methy lthienyl)acryloyl adducts with papain, cathepsins B and L, and two oxy anion hole mutants of cathepsin B (Q23S and Q23A) show that extensive polarization throughout the pi-electron chain occurs for the bound acy l group in the active sites. A similar result is obtained using the sp ecific chromophoric substrate ethyl L-phenylalanyl)amino]-3-(5-methylt hienyl)acrylate. By using C-13=O substitution it is possible to detect the acyl C=O stretching frequency, v(C=O), for each acyl enzyme. A co rrelation between v(C=O) and log k(3), where k(3) is the deacylation r ate constant, is found where v(C=O) increases with increasing reactivi ty. This is exactly the opposite sense to the relationship found for a series of acyl serine proteases [Carey & Tonge (1995) Ace. Chem. Res. 28, 8], The opposite trend in the direction of the correlation for th e acyl cysteine proteases is ascribed to the strong electron polarizin g forces in the active site, due principally to the active-site alpha- helix dipole, giving rise to canonical (valence bond) forms of the acy l group which change the hybridization about the C=O carbon atom, A co rrelation is also observed between the absorption maximum, lambda(max) , and log k(3) for each acyl cysteine protease. As the deacylation rat e increases, 214-fold across the series, lambda(max) red-shifts from 3 67 to 384 nm. It is proposed that differential interactions between th e active site's alpha-helix dipole and the acyl chromophore give rise to the observed changes in lambda(max), with the red shift being cause d principally by interactions with the excited electronic state, which has a high degree of charge separation, and the dipole, Similar inter actions between the dipole and the resembles the transition state, are proposed as the negatively charged tetrahedral intermediate, which so urce of differential rates in deacylation, It is interesting to note t hat similar energies are operating in both cases. A shift in lambda(ma x) from 367 to 384 nm corresponds to a change in electronic absorption transition energies of 3.2 kcal/mol and a change of deacylation rate constants of 214-fold also corresponds to a change of activation energ ies of 3.2 kcal/mol.